Cathepsin D Isozymes from Porcine Spleens

Six cathepsin D isozymes have been purified from porcine spleen using a large scale purification proce- dure. Five isozymes, I to V, have an identical molecular weight of 50,000 and are similar in specific activity. Isozymes I to IV contained two polypeptide chains each. The light and heavy chains have M, = 15,000 and 35,000, respectively. Isozyme V is a single polypeptide. The molecular weight of the sixth isozyme is about 100,000 and it has only 5% of the specific activity of the other isozymes. On Ouchterlony immunodiffusion, an anti- serum formed precipitin lines against the urea-dena- tured isozyme with M,. = 100,000. This immunoreactiv- ity showed immunoidentity with those formed against other isozymes. The NHz-terminal sequence of light chains was iden- tical for the isozymes. This sequence is homologous to the NHz-terminal sequence of other acid proteases, es- pecially near the region of the active center aspartate- 32. The NHz-terminal sequence of the single chain, iso- zyme V, is apparently the same as the light chain se- quence. The NHz-terminal sequence analysis of the heavy chain from isozyme I produced two sets of re- lated sequences, suggesting the presence of structural microheterogeneity. The carbohydrate analysis of the isozymes, the light chain, and the heavy chain revealed the presence of possibly four attachment sites, with one in the light chain and three in the heavy chain. Each carbohydrate unit contains 2 residues of mannose and 1 residue of glucosamine. The results suggest that the high molecular weight cathepsin D (M,. = 100,000) is the probable precursor of the single chain (Mr = 50,000), which in turn produces the two-chain isozymes. These are likely in uiuo proc- esses. Cathepsin D (EC.3.4.23.5) is a lysosomal acid protease widely distributed in eukaryotic and prokaryotic cells. There is increasing evidence that this enzyme and cathepsin B are the main lysosomal endopeptidases responsible for intracel- lular protein hydrolysis (1, 2). Since the name cathepsin D